Summary of Work: Infection by the HIV virus causes a change in a number of physiological processes, the etiologies of which are poorly understood. The initial step is syncitium formation, currently accepted as involving HIV gp120 and gp41, CD4 and a chemokine receptor. Later developments include the depletion of T cells expressing CD4 and B cells expressing immunoglobulins, and changes in the regulation of cytokines. Crucial to an understanding of these processes and to developing therapeutic strategies related to these changes is the determination of the structural motifs critical to the physiological processes involved in the changes. We are employing the methodologies we have been using for epitope determination (protection assays and surface modification reactions combined with mass spectrometry) to probe receptor-ligand pairs relevant to HIV infection including: a) CD4 and gp120 and; b) the complex between gp120, CD4 and chemokine receptor CXCR4. The ternary complex between gp120, CD4 and a chemokine receptor is now accepted as the crucial interaction involved in cellular infection by the HIV. Identification of the sites of interaction on any or all of the molecules involved in the complex will provide information that should be useful in design of vaccines and/or drugs that inhibit infection. Our initial experiments have been to probe uncomplexed gp120 in solution using a variety of enzymes to characterize the digestion products. Included in this part of the project is the determination of the the glycosylation pattern of gp120. We have found this to be extremely complex with individual consensus glycosylation sites being non-glycosylated, glycosylated with high mannose structures and glycosylated with complex structures. At the same time as these experiments are being carried out we are probing gp120/CD4 interaction site using a new strategy based on the specific labeling of the protein interaction site using a novel cleavable fluorescent crosslinker. The non-covalent complex is formed under non-denaturing conditions and then coupled with the crosslinker. The crosslinked complex has been isolated and cleaved into the component proteins, now carrying fluorescent tags. Characterization of the residues modified by the crosslinker is currently in progress. In a similar study, the sites of interaction between HIV-integrase and two inhibitors is being studied by photoaffinity labeling and MS peptide mapping in collaboration with the NCI group.